Fibrillar collagens are structural proteins that assemble into a complex ordered structure of molecules cross-linked together to form an interconnected supramolecular fibril structure (Ottani et al. 2002). Collagens contribute to the mechanical properties of almost all tissues throughout the body including skin, tendon, ligament, bone, and cartilage (Bailey, Paul, and Knott 1998; Ottani et al. 2002).
Collagen is highly resistant to enzymatic breakdown, but is susceptible to a small number of specialized collagenolytic enzymes or collagenases. In part due to this resistance to enzymatic cleavage, collagen has a very slow turnover rate in many tissues of the body. The half-life for collagen has been reported on the order of decades in healthy tissues (Verzijl, DeGroot, Thorpe et al. 2000; Maroudas, Palla, and Gilav 1992; Bank et al. 1999). Due to the long protein half-life in vivo, collagen is one of the proteins that undergo spontaneous glycation and the formation of measurable amounts of Advanced Glycation Endproducts (AGEs) during aging (Choudhary et al. 2011; Sell and Monnier 2004; Verzijl, DeGroot, Oldehinkel et al. 2000; Verzijl, DeGroot, Thorpe et al. 2000).
Glycation, also called non-enzymatic glycosylation, is a spontaneous, non-enzymatic process in which a reducing sugar, such as glucose or fructose, reacts with a free amino group (e.g. lysine or arginine) to form a reactive Schiff base. The Schiff base then rearranges to form an Amadori product which undergoes further reactions, collectively known as a Maillard reaction, to form AGEs (Aronson 2003; Bailey, Paul, and Knott 1998). Of interest is that these reactions can result in stable covalent cross-links between two amine groups of amino acids, such as lysine or arginine (Bailey, Paul, and Knott 1998; Sell and Monnier 2004; Verzijl et al. 2002).
AGE accumulation in soft tissues is a function of tissue aging and accelerated by diabetes due to hyperglycemia (Bai et al. 1992; Freemont and Hoyland 2007; Reddy 2003; Reddy, Stehno-Bittel, and Enwemeka 2002). AGE cross-linking results in changes in the mechanical properties of soft tissues, which include increased Young's modulus, maximum failure load, stress, and toughness, as well as decreased elongation and strain, while in mineralized tissue there are minimal changes in these properties after glycation (Reddy 2003; Reddy, Stehno-Bittel, and Enwemeka 2000). In addition, AGE cross-linking has been implicated in a variety of pathological aging-related changes, including vascular (Aronson 2003), and articular cartilage stiffening (Chen et al. 2002; Verzijl, DeGroot, Oldehinkel et al. 2000), which may contribute to arteriosclerosis and osteoarthritis, respectively.
Computational molecular modeling was previously performed using steered molecular dynamics to simulate mechanical loading of a covalently cross-linked collagen (Bourne and Torzilli 2011). These loading conditions approximated mechanical force transmitted through covalent intermolecular cross-links, such as those caused by AGEs. Computational results predicted that force transmitted via cross-links would result in local disruption and micro-unfolding of the collagen triple helix at approximately 350 pN (minor micro-unfolding) and 900 pN (major micro-unfolding) (Bourne and Torzilli 2011). These values are well below those previously described as causing collagen failure (Bourne and Torzilli 2011; Buehler 2006; Tang et al. 2010).
Based upon molecular modeling and the experimental results set forth herein, it has been shown that mechanical forces on cross-linked collagen substrates would accelerate enzyme degradation, and that it would be desirable to decrease or eliminate these cross-links, such as those caused by AGE, prior to implantation of grafts, implants, scaffolds, or constructs into a subject.